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Ganoderma Lucidum Causes Apoptosis in Leukemia, Lymphoma and Multiple Myeloma Cells

Ganoderma Lucidum Causes Apoptosis in Leukemia, Lymphoma and Multiple Myeloma Cells

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Leukemia Research 30 (2006) 841–848
Ganoderma lucidum
causes apoptosis in leukemia,lymphoma and multiple myeloma cells
Claudia I. M¨uller
, Takashi Kumagai
, James O’Kelly
,Navindra P. Seeram
, David Heber
, H. Phillip Koeffler
Cedars-Sinai Medical Center, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States
 Division of Haematology/Oncology, University Hospital, Freiburg, Germany
Center for Human Nutrition, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States
Received 4 May 2005; received in revised form 5 December 2005; accepted 6 December 2005Available online 19 January 2006
Over many centuries, herbal remedies have treated a variety of ailments. This empiric observational approach has produced a numberof leads for formulated medicines.
Ganoderma lucidum
extract was screened for its anti-proliferative activity using a panel of 26 humancancer cell lines. The six most sensitive hematologic cell lines were: HL-60 (ED50 26
g/ml), U937 (63
g/ml), K562 (50
g/ml), Blin-1(38
g/ml).CellcycleanalysesrevealedaG2/Marrest,mostprominentlyinHL-60cells.Fourhematopoietic cell lines (HL-60, Blin-1, U937, RPMI8226) were examined for apoptosis, which ranged between 21 and 92%. After exposureto
G. lucidum
extract, HL-60 cells became multinucleated with an increased DNA content. These results indicate that
G. lucidum
extracthas a profound activity against leukemia, lymphoma and multiple myeloma cells and may be a novel adjunctive therapy for the treatment of hematologic malignancies.© 2005 Elsevier Ltd. All rights reserved.
Keywords: Ganoderma lucidum
; Leukemia; Lymphoma; Multiple myeloma; Apoptosis; Growth arrest
1. Introduction
(Fr.)Karst(commonnames:Reishei,Lingzhi) is a herbal mushroom that has been used for cen-turies in Traditional Chinese Medicine (TCM) for its healthpromotingproperties[1].Thefruitingbodies,sporesandcul- tivated mycelia of 
G. lucidum
as well as its extracts are usedworld-wide as ingredients in health foods, herbal medicinesand dietary supplements (extracts and powdered forms) andhave been used as anti-cancer agents and for prevention andtreatment of various other diseases in ancient China (100
Corresponding author at: Division of Hematology/Oncology, DavisBuilding 5065, Cedars-Sinai Medical Center, David Geffen School of Medicine at UCLA, 8700 Beverly Boulevard, Los Angeles, CA 90048,United States. Tel.: +1 310 423 7759; fax: +1 310 423 0225.
 E-mail address:
MullerCI@cshs.org (C.I. M¨uller).
ited growth of several cancer cell lines including the humanprostate PC-3 cancer cells, and several human bladder can-cer cell lines. These cells were arrested in the G2/M phaseof their cell cycle[7,8].Also,
in a dose- andtime-dependent manner inhibited the cell proliferation andinduced apoptosis of HT-29, a human colon carcinoma cellline and MDA-MB-231 and MCF-7 breast cancer cell lines[5,9,10].The phytochemical constituents of 
includepolysaccharides, proteins, nucleosides, fatty acids, sterols,cerebrosides and triterpenes[11].The triterpenoids com- mon to
include the ganoderic acids that may beconsidered as a chemical marker compound to authenticateand/orstandardize
extractsandproducts[12,13].The polysaccharide fraction of 
was shownto slow growth of sarcoma cells growing in mice[14].The polysaccharides were able to induce the expression of IL-1, IL-6, IL-12, IFN-
, TNF-
0145-2126/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.leukres.2005.12.004
C.I. M¨ uller et al. / Leukemia Research 30 (2006) 841–848
monocytes–macrophages and T lymphocytes which might,in part, mediate some of their anti-tumor activity[1,15,16].Furthermore, these polysaccharides may also have immuneenhancingpropertiesperhapsasaconsequenceoftheirabilityto stimulate cytokine production[15].Triterpenes have been shown to inhibit growth of humanhepatomaHuh-7cellsassociatedwithG2cellcyclearrest,butthecompoundshadnoeffectongrowthof“normal”livercelllines in vitro[2].Moreover, the triterpene-fraction of 
inhibited the primary and metastatic tumor growth of Lewis lung carcinoma (LLC) cells implanted in mice[17].This study investigated the anti-cancer effects of 
usingapanelof26humancancercelllines(16hema-tologic malignancies; see Section2).A detailed description of the characteristics of each cell line is also given inTable 1.
Table 1Humancancercelllinesexaminedforanti-proliferativeeffectsof 
extractCell line Characteristics
Acute myeloblastic leukemia(FAB M2)16 hematologicalcell lines
Diffuse histiocytic lymphoma(monocytic leukemia cells)NB4 Promyelocytic leukemia (FABM3)THP-1 Acute monocytic leukemia (FABM5)
Erythroid chronic myeloidleukemia (blast crisis)
Pre-B acute lymphoblasticleukemia
Non-T, non-B acutelymphoblastic leukemiaJurkat T-cell acute lymphoblasticleukemia
Multiple myeloma (IgG
)ARH77 Multiple myeloma (IgG
)U266 Multiple myeloma (IgE
)NCI-H929 Multiple myeloma (IgA
)Daudi Burkitts lymphomaRamos Burkitts lymphomaNCEB-1 Centroblastic–centrocytic, diffuselymphomaSUDHL6 Diffuse large-B-cell lymphomaLNCaP Prostate cancer (androgenreceptor positive)10 solid tumorcell linesPC-3 Prostate cancer (androgenreceptor negative)DU145 Prostate cancer (androgenreceptor negative)MCF-7 Breast cancer (estrogen receptorpositive)MDA-MB231 Breast cancer (estrogen receptornegative)HT29 Colorectal cancerPANCI Pancreatic cancerASPC1 Pancreatic cancerBxPC-3 Pancreatic cancerNCI-H520 Non-small cell lung cancer(squamous cell carcinoma)The six most sensitive cell lines are in bold.
2. Materials and methods
2.1. Extraction of G. lucidum and standardization by HPLC analysisG. lucidum
fruiting bodies were collected and authen-ticated in China by Phytomedical Research Inc. (Beijing,China), and Botanica Biosciences (Ojai, CA). A commercialstandard of ganoderic acid C2 was purchased from Chro-madex (Santa Ana, CA).
G. lucidum
fruiting bodies wereextracted in water and lyophilized to yield a dark brownpowder.All solvents were HPLC (high performance liquid chro-matography) grade and were purchased from Fisher (Tustin,CA).
extract (22.28mg) was sonicated for30min with 4ml (MeOH:water, 1:1 v/v). Ganoderic acid C2was dissolved in MeOH to make a stock solution of 1mg/mlthat was further serially diluted with MeOH:water (1:1 v/v).Quantitationwasdonebypeakareameasurementsincompar-ison with a standard curve generated for ganoderic acid C2.All samples (25
l injection volume) were filtered (0.22
m)before analysis on a Waters 2690 HPLC system equippedwitha996PDAdetector(Waters,Milford,MA).Compoundswereelutedat1.00ml/minonaNovapak(Waters)RP-18col-umn (150mm
3.9mm i.d., 5
m) with a Symmetry C18guard column (20mm
3.9mm i.d., 5
m). The eluent con-sisted of 2% aqueous acetic acid (A) and acetonitrile (B). Agradient solvent system was used as follows: 0–5min 95% Ain B; 6–60min 5% A in B. The wavelength of detection was254nm.
2.2. Cell lines
Cell lines that were studied included: myeloid leukemia(HL-60, U937, K562, THP-1, NB4), acute lymphoblasticleukemia (B- and T-ALL) (Blin-1, Nalm-6, Jurkat), mul-tiple myeloma (RPMI8226, ARH77, U266, NCI-H929),Burkitt’s lymphoma (Daudi, Ramos), non-Hodgkin’s lym-phoma (NCEB-1, SUDHL6), prostate cancer (LNCaP, PC-3,DU145), breast cancer (MCF-7, MDA-MB-231), colorectalcancer (HT-29), pancreatic cancer (PANCI, ASPC1, BxPC-3)andnon-smallcelllungcancer(NSCLC)(NCI-H520)(seeTable 1).Most cell lines were obtained from American TypeCulture Collection (ATCC, Rockville, MD), Blin-1, NCEB-1 and SUDHL6 cells were generously provided by Sven deVos(UniversityofCalifornia,LosAngeles).NB4cellswereakind gift from M. Lanotte (St. Louis Hospital, Paris, France).Cells were maintained in culture according to recommenda-tions.
2.3. MTT assays
An initial screening was done to explore the anti-proliferative effects of the herbal extract on the 26 humancancer cell lines. Cells (3
) were cultured inthe presence of 50 and 100
G. lucidum
extract for
C.I. M¨ uller et al. / Leukemia Research 30 (2006) 841–848
96h in 96-well plates (Flow Laboratories, Irvine, CA).Thereafter, 10
l of MTT solution (in 5mg/ml PBS,Roche) was added to each well and incubated for 4hin a humidified atmosphere at 37
C according to themanufacturer’s protocol. Consecutively, 50
l solubilizationsolution (20% SDS) was added into the wells and incubatedovernight (16h). After culture, cell number and viabilitywere evaluated by measuring the mitochondrial-dependentconversion of the yellow tetrazolium salt MTT to purpleformazan crystals by metabolic active cells. The resultingcolored solution was quantified at 540nm using an enzyme-linked immunoabsorbent assay reader (ELISA reader,Bio-Rad).
2.4. Cell cycle analysis
Cells (3
) were incubated for 72h either withor without
G. lucidum
g/ml). They were collected,washed, suspended in cold 1
PBS, fixed in 75% methanoland stained with propidium iodide (PI). Cell cycle analysiswas performed using the Becton Dickinson Flow Cytometer.
2.5. Assessment of apoptosis by Annexin V assay
Apoptotic cell death was examined by Annexin V-apoptosis detection kit (Pharmingen Inc., San Diego, CA).During early stages of apoptosis, phosphatidylserine (PS)becomes externalized on the outer plasma membrane. Inorder to be able to distinguish between apoptosis andnecrosis, cells were stained with FITC-labeled AnnexinV and propidium iodide (PI). Annexin V binds to theexternalized PS, whereas PI is able to penetrate theincreasingly permeable plasma membrane during necrosisor later stages of apoptosis and binds to cellular DNA.Cells were analyzed by fluorescence activated cell sorting(FACS).Four hematological cell lines (HL-60, U937, Blin-1 andRPMI8226) were treated with
G. lucidum
at four differentconcentrations(50,100,150and200
g/ml)for72h.Controlcells and treated cells were analyzed for staining by AnnexinV and PI.
2.6. Assessment of apoptosis by determination of mitochondrial membrane potential
Apoptosis was further investigated by analysis of the mitochondrial membrane potential (
) by JC-1assay according to the manufacturers protocol (CellTechnology, MN). JC-1 (5,5
-tetraethylbenzimidazolcarbocyanine iodide) is a lipophiliccationic dye which selectively incorporates into intactmitochondria and undergoes a reversible change in flu-orescence emission from green to red as mitochondrialmembrane potential increases. During apoptosis, loss of themitochondrial membrane potential occurs. In cells with highmembrane potential, the formation of dimers of the dye ispromoted, resulting in red fluorescence. In contrast, in cellswithlowmembranepotential,JC-1isinitsmonomericform,which then fluoresces green. The red/green fluorescenceintensity ratio is used to analyze whether a cell is apoptoticor not. A decrease in red/green ratio indicates an increasein apoptotic cells. Fluorescence of the cells is measuredby flow cytometry. Formation of JC-1 aggregates withinthe mitochondria results in emission of red fluorescencein the 590nm spectrum. If the mitochondriae collapseduring apoptosis, the fluorescence shifts to the green 510nmspectrum.
2.7. Western blot analysis
Total cell lysate (25
g) was electrophoresed on 10–20%SDS-polyacrylamide gel (Bio-Rad, Hercules, CA) andtransferred by electroblotting to a polyvinylidene fluoridemembrane (Pall Corporation, Pensacola, FL). The mem-brane was incubated overnight with the following anti-bodies: anti-p21
(SC-397, 1:500 dilution) and anti-p27
(SC-528, 1:500 dilution), both rabbit polyclonalantibodies from Santa Cruz Biotechnology (Santa Cruz,CA) followed by a secondary horseradish peroxidase-conjugated donkey anti-rabbit antibody (Amersham Bio-sciences). Detection was performed using the SuperSignal
chemiluminescence substrate (Pierce, Rockford, IL). Blotswere also subjected to overnight incubation with amurine monoclonal GAPDH antibody (RDI-TRK5G4-6C5, 1:10,000 dilution; Research Diagnostics, Flanders,NJ) followed by a secondary horseradish peroxidase-conjugated sheep anti-mouse antibody (Amersham Bio-sciences). Western blots were stripped between hybridiza-tionswithstrippingbuffer(10mMTris–HClpH2.3,150mMNaCl).
2.8. Cytospin preparations and staining
Cytospin preparations were performed for seven hemato-logic(NCI-H929,RPMI8226,Blin-1,Nalm-6,Daudi,U937,HL-60) and six solid tumor (ARO, BHP2-7, PC3, DU145,NCEB-1, MCF-7) cell lines after 96h treatment with
g/ml). Slides were consecutively stained withDiff Quik 
(Dade Behring, Switzerland) staining solutionaccording to the manufacturer’s protocol.
3. Results
3.1. HPLC analysis of G. lucidum extract 
G. lucidum
extract was standardized to 0.15% gan-oderic acid C2 content (Fig. 1a).Fig. 1bshows the HPLC chromatogram of ganoderic acid C2 standard eluting at aretention time of 32.9min.Fig. 1cshows the
G. lucidum
extract spiked with ganoderic acid C2 standard confirmingits presence at the retention time of 32.9min.

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